Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocopetentent rats

AIM: To characterize the host response to hepatitis B virus (HBV) infection in human hepatocytes transplanted into immunocompetent rodent rats tolerized by, and transplanted with primary human hepatocytes. METHODS: One week after the transplantation, rats were inoculated with HBV, and viral gene expression, replication, and host response was monitored. RESULTS: HBV DNA was detectable in serum for at least 60 days. HBsAg levels rose steadily for 3 weeks post-inoculation and then plateaued at a level of about 0.6 pg/ml. HBV RNA was also found in liver at levels that remained constant through the time course. Immunofluorescence revealed clusters of hepatocytes that stained positive for HBcAg. The presence of HBV covalently closed circular DNA (cccDNA) in liver was demonstrated using nuclease digestion of single-stranded DNA followed by PCR. Serum ALT levels rose and reached a peak level of 180 IU/L on day 18, but remained elevated for 60 days. Histology revealed a progressive predominantly mononuclear lobular hepatitis. CONCLUSION: These data indicate that human hepatocytes transplanted into rats rendered tolerant to these cells, when infected by HBV, results in biochemical as well as histological evidence of hepatitis that accompanies viral gene expression, and DNA replication.     

Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ

Immune tolerance of human cells without generalized immunosuppression was created in groups of normal fetal rats at 17 days of gestation by inoculation ip with primary human hepatocytes in utero. One day after birth, suspensions of human hepatocytes were transplanted via intrasplenic injection and one week later groups of rats were inoculated with hepatitis B virus (HBV). Tolerized rats that were transplanted with human hepatocytes and subsequently infected with HBV produced hepatitis B surface antigen (HBsAg) in serum beginning on day 3. Levels rose fivefold and remained stable at 0.75 pg/ml through at least 60 days. Of cells that stained positive for human serum albumin, approximately 30% were found to be also positive for HBsAg by immunohistochemistry. Serum HBV DNA was detectable from 1 to 15 weeks postinfection. Finally, covalently closed circular DNA, reflecting HBV replication, was found in liver and serum. Controls that were tolerized and not transplanted, but inoculated with HBV, as well as untreated controls, had no evidence of HBV gene expression or replication under identical conditions. The data support the conclusion that primary human hepatocytes transplanted into genetically immunocompetent rodent hosts, survive and maintain sufficient differentiation to produce human serum albumin and be infected by HBV.     

Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocompetent rats

AIM: To characterize the host response to hepatitis B virus (HBV) infection in human hepatocytes transplanted into immunocompetent rodent rats tolerized by, and transplanted with primary human hepatocytes.METHODS: One week after the transplantation, rats were inoculated with HBV, and viral gene expression, replication,and host response was monitored.RESULTS: HBV DNA was detectable in serum for at least 60 days. HBsAg levels rose steadily for 3 weeks postinoculation and then plateaued at a level of about 0.6 pg/mi. HBV RNA was also found in liver at levels that remained constant through the time course. Immunofluorescence revealed clusters of hepatocytes that stained positive for HBcAg. The presence of HBV covalently closed circular DNA (cccDNA) in liver was demonstrated using nuclease digestion of single-stranded DNA followed by PCR. Serum ALT levels rose and reached a peak level of 180 IU/L on day 18, but remained elevated for 60 days. Histology revealed a progressive predominantly mononuclear lobular hepatitis.CONCLUSION: These data indicate that human hepatocytestransplanted into rats rendered tolerant to these cells, when infected by HBV, results in biochemical as well as histological evidence of hepatitis that accompanies viral gene expression,and DNA replication.     

Peginterferon-alpha-2a (40KD) and ribavirin for 16 or 24 weeks in patients with genotype 2 or 3 chronic hepatitis C

BACKGROUND & AIMS: Standard therapy of patients with chronic hepatitis C virus (HCV) infected with HCV genotype-2 or -3 is the combination of pegylated interferon-alpha and ribavirin for 24 weeks. Whether shorter treatment durations are possible for these patients without compromising sustained virologic response rates is unknown. METHODS: Patients chronically infected with HCV-2 (n = 39), HCV-2/3 (n = 1), or HCV-3 (n = 113) were treated with peginterferon-alpha-2a (180 microg/wk) plus ribavirin 800-1200 mg/day. HCV RNA was quantitatively assessed after 4 weeks. Patients with a rapid virologic response (HCV RNA below 600 IU/mL) were randomized for a total treatment duration of 16 (group A) or 24 weeks (group B). All patients with HCV RNA > or =600 IU/mL at week 4 (group C) were treated for 24 weeks. End-of-treatment and sustained virologic response were assessed by qualitative RT-PCR (sensitivity 50 IU/mL). RESULTS: Only 11 of 153 patients (7%) were allocated to group C. End-of-treatment and sustained virologic response rates were 94% and 82%, (group A), 85% and 80% (group B), and 73% and 36% (group C), respectively. In patients infected with genotype HCV-3 and high viral load (>800,000 IU/mL), a significant lower sustained virologic response rate was found than in patients infected with HCV-3 and a viral load lower or equal to 800,000 IU/mL (59% vs 85%, respectively; P = .003). CONCLUSIONS: In HCV-2 and -3 (low viral load)-infected patients who have a rapid virologic response, treatment for 16 weeks with peginterferon-alpha-2a and ribavirin is sufficient. In patients infected by HCV-3 (high viral load), longer treatment may be necessary.     

Divergent activities of interferon-alpha subtypes against intracellular hepatitis C virus replication.

BACKGROUNDS: Interferon (IFN)-alpha is represented by several structurally related subtypes that show different antiviral and anti-tumor effects. Here, we analyzed differential effects of IFN-alpha subtypes on intracellular hepatitis C virus (HCV) replication using HCV subgenomic replicon system as a model. METHODS: Huh7 and HeLa cells supporting expression of HCV replicon were treated with various concentrations of five recombinant human IFN-alpha subtypes 1, 2, 5, 8, and 10, and with IFN-alpha con1. The effects of IFNs on various cell-signaling pathways were assayed by using ISRE-, GAS-, AP1-, NF-kappa B-, CRE-, and SRE-luciferase reporter plasmids. RESULTS: Each IFN-alpha subtype suppressed HCV replication in a dose-dependent manner. Among them, IFN-alpha8 was the most effective, while IFN-alpha1 was the least effective with 50% inhibitory concentrations of 0.123IU/ml versus 0.375IU/ml, respectively. These differential effects against HCV replication did not correlate with levels of the IFN-responsive ISRE or GAS reporter activities, nor they did activate the other reporters, AP1, NF-kappa B, CRE and SRE. CONCLUSION: There were divergent effects of IFN-alpha subtypes against HCV replication that may be through JAK-STAT-independent pathways. Exploring further mechanisms of action may elucidate IFN-mediated cellular antiviral mechanisms.     

An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain

Aim: The hepatitis C virus (HCV) strain JFH‐1 was cloned from a patient with fulminant hepatitis. A JFH‐1 subgenomic replicon and full‐length JFH‐1 RNA efficiently replicate in cultured cells. In this study, an infectious, selectable HCV replicon containing full‐length JFH‐1 cDNA was constructed. Methods: The full‐genome replicon was constructed using the neomycin‐resistant gene, EMCV IRES and wild‐type JFH‐1 cDNA. Huh7 cells were transfected with RNA synthesized in vitro, and then cultured with G418. Independent colonies were cloned to establish cell lines that replicate the full‐length HCV replicon. Results: HCV RNA replication was detected in each isolated cell line. HCV proteins and HCV RNA were secreted into culture medium, and exhibited identical density profiles. Interestingly, culture supernatants of the replicon cells were infectious for naïve Huh7 cells. Long‐term culture did not affect replication of replicon RNA in the replicon cells, but it reduced core protein secretion and infectivity of culture supernatant. Culture supernatant obtained after serial passage of replicon virus was infectious for Huh7 cells. Conclusions: Selectable infection was established using HCV replicon containing full‐length genotype 2a JFH‐1 cDNA. This system might be useful for HCV research.     

Griseofulvin, an oral antifungal agent, suppresses hepatitis C virus replication in vitro

Aim: Hepatitis C virus (HCV), which infects an estimated 170 million people worldwide, is a major cause of chronic liver disease. The current standard therapy for chronic hepatitis C is based on pegylated interferon (IFN)alpha in combination with ribavirin. However, the success rate remains at approximately 50%. Therefore, alternative agents are needed for the treatment of HCV infection. Methods: Using an HCV-1b subgenomic replicon cell culture system (Huh7/Rep-Feo), we found that griseofulvin, an oral antifungal agent, suppressed HCV-RNA replication and protein expression in a dose-dependent manner. We also found that griseofulvin suppressed the replication of infectious HCV JFH-1. A combination of IFNalpha and griseofulvin exhibited a synergistic inhibitory effect in Huh7/Rep-Feo cells. Results: We found that griseofulvin blocked the cell cycle at the G(2)/M phase in the HCV subgenomic replicon cells, but did not inhibit HCV internal ribosome entry site-dependent translation. Conclusion: Our results suggest that griseofulvin may represent a new approach to the development of a novel therapy for HCV infection.     

Mathematical modeling of primary hepatitis C infection: noncytolytic clearance and early blockage of virion production

BACKGROUND & AIMS: Although hepatitis C virus kinetics and immune determinants during primary infection have been described, the virus-host interplay is not fully understood. We used mathematical modeling to elucidate and quantify virus-host dynamics. METHODS: Ten chimpanzees were infected intrahepatically with H77-RNA (n = 3) or intravenously with infected serum. Blood samples were taken 1-3 times per week for 6 months. A new model was fitted to the observed HCV RNA and alanine aminotransferase (ALT) kinetics. RESULTS: After infection, viral levels increased in a biphasic manner with a transient decline in between. This can be explained by a partial block (mean, 91%) of virion production, possibly due to an endogenous type I interferon response. After reaching maximum levels, a long viral plateau (mean, 6.1 log cp/mL) can be explained by blind homeostasis and lack of susceptible cells. Modest elevations in ALT levels (21-93 IU/L) were concurrently observed, indicating a shorter half-life of infected versus noninfected hepatocytes (mean ratio, 2.6). Following the ALT flare, viral titers rapidly declined to a lower (mean, 4.5 log cp/mL; n = 6) or undetectable level (n = 4). This decline is compatible with increased cell death (mean minimal estimate half-life, 28.7 days) and noncytolytic clearance (mean maximal estimate half-life, 24.1 days) of infected cells. CONCLUSIONS: Our results quantify virus-host dynamics during primary HCV infection and suggest that endogenous type I interferon slows virus production in the early acute phase. Partial or effective virus control correlates with the half-life of infected cells regulated by both cytolytic and noncytolytic mechanisms.     

Pegylated arginine deiminase lowers hepatitis C viral titers and inhibits nitric oxide synthesis

BACKGROUND: The arginine-degrading enzyme, arginine deiminase conjugated to polyethylene glycol (ADI-SS PEG 20,000 mw), reduces extracellular arginine, has minimal toxicity, decreases tumor burden and improves liver function in patients with chronic hepatitis C virus infection (HCV) and inoperable hepatocellular carcinoma (HCC). Reduced extracellular arginine inhibits viral replication through unknown mechanisms. It is hypothesized that ADI-SS PEG 20,000 mw reduces HCV viral titers through nitric oxide (NO)-dependent effects. METHODS: The effects of ADI-SS PEG 20,000 mw (dose, 160 IU/m2; three cycles of four once-weekly i.m. injections) on HCV titers, serum NO and plasma arginine, were evaluated using archived plasma from patients with HCC and HCV and in vitro cell model measurements of HCV replication. RESULTS: ADI-SS PEG 20,000 mw selectively inhibited HCV replication in vitro (IC50 = 0.027 IU/mL). Fifteen HCC/HCV patients completed treatment. The HCV titers were reduced by up to 99% in five out of 10 (50%) HCV-serotype 1b patients (P = 0.0093). These patients also experienced significant improvements in liver function (P = 0.0091). There were concomitant reductions of plasma arginine and serum NO levels. The HCV titer was not reduced in HCV-type 2c patients. CONCLUSION: Reduction of extracellular arginine by ADI-SS PEG 20,000 mw in HCC patients reduces HCV viral titers and improves liver function, possibly through suppression of NO.     

Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were trans...     

Hepatitis C virus replication in Caucasian HIV controllers

Summary. Whether HIV controllers, patients who spontaneously control HIV viraemia, are able to control hepatitis C virus (HCV) infection, in terms of spontaneous clearance or lower HCV replication, is not well understood. To assess to what extent Caucasian HIV controllers are able to control HCV replication and potential associated factors, plasma HIV‐1 and HCV RNA levels, anti‐HCV antibodies, HCV genotype and human leucocyte antigens (HLA) typing were determined in samples from 75 HIV controllers (33 viraemic controllers, <1000 HIV‐1 RNA copies/mL, and 42 elite controllers, <40 HIV‐1 RNA copies/mL) and compared with 261 HIV‐infected noncontrollers. We did not find differences in the HCV spontaneous clearance rates between groups. However, we interestingly found a lower HCV viral load in HIV controllers, alongside a different distribution of HCV genotypes in relation to the comparison group. In addition, HLA‐B57 was associated with a lower HCV viral load in the control group and HIV controllers, and conversely, HLA‐B35 with higher HCV viral load in HIV controllers. The subrepresentation of HCV genotype 1 and the overrepresentation of HLA‐B57 only partly explained the lower HCV viral load found in HIV controllers. In fact, HIV controller status was independently associated with lower HCV viral load, together with HCV genotype non‐1, the presence of HLA‐B57 and absence of HLA‐B35. Caucasian HIV controllers are able to better control HCV replication, in terms of lower HCV viral load levels. These findings support the idea that some common host mechanisms are involved in the defence against these two persistent infections.     

Viral clearance occurs very early during the natural resolution of hepatitis C virus infection in persons with haemophilia

We studied spontaneous hepatitis C virus (HCV) RNA clearance in 12 haemophilic patients. In their earliest anti-HCV positive samples, HCV RNA was undetectable in eight patients (66%), positive by polymerase chain reaction (PCR) but negative by branched-DNA (bDNA) in three others, and quantifiable by bDNA (4839 IU/mL) in only one patient. In contrast, in earliest anti-HCV positive samples from eight matched controls who had persistent viremia, HCV RNA was quantifiable by bDNA in seven (P = 0.0008) and at higher levels (range 4644-678 515 IU/mL; median 43 532 IU/mL). From initial HCV infection, HCV RNA cleared in 7 months or less in four patients and in 1-2 years in six others. HCV persisted for 5 years before clearance in the absence of repeated exposure in one patient. We conclude that HCV clearance usually but not always occurs within 1-2 years after infection and is more likely in those with lower than in those with higher early viral loads.     

Gene expression profile of Huh‐7 cells expressing hepatitis C virus genotype 1b or 3a core proteins

Background: The liver disease expression in chronic hepatitis C patients is variable and may partially depend on the sequence of the infecting viral genotype. Aim: To identify some hepatitis C virus (HCV) genotype‐specific virus–host interactions potentially leading to clinically significant consequences. Methods: We compared the gene expression profile of Huh‐7 cells transiently expressing the core protein of HCV genotype 1b and 3a using microarray technology. Results: Thirty‐two genes were overexpressed in Huh‐7 transfected with the HCV genotype 1b core protein and 57 genes in cells transfected with the genotype 3a core protein. On the other hand, we found 20 genes downregulated by core 1b and 31 genes by core 3a. These included genes involved in lipid transport and metabolism, cell cycle, immune response and insulin signalling. Conclusion: The expression of HCV core proteins of different genotypes leads to a specific gene expression profile. This may account for the variable disease expression associated with HCV infection.     

A cell-based, high-throughput screen for small molecule regulators of hepatitis C virus replication

BACKGROUND & AIMS: Only half of patients with chronic hepatitis C virus (HCV) infection experience sustained virologic response to pegylated-interferon and ribavirin, which cause numerous side effects. Thus, the identification of more effective and better tolerated agents is a high priority. We applied chemical biology to screen small molecules that regulate HCV. METHODS: We first optimized the Huh7/Rep-Feo replicon cell line for the 384-well microplate format and used this line to screen a large library of well-characterized, known biologically active compounds using automated technology. After identifying several molecules capable of either stimulating or inhibiting HCV replication in this primary screen, we then validated our hit compounds using a full-length HCV replicon cell line in secondary screens. RESULTS: We identified and validated a number of antiviral and proviral agents, including HMG-CoA reductase inhibitors (antiviral) and corticosteroids (proviral). The finding of increased replication associated with corticosteroids suggests that these agents directly promote viral replication independent of their suppressive effects on the immune response. The finding of antiviral activity associated with the HMG-CoA reductase inhibitors implies an important role for lipid metabolism in the viral life cycle. CONCLUSIONS: We have developed a simple, reproducible, and reliable cell-based high-throughput screening assay system using an HCV replicon model to identify small molecules that regulate HCV replication. This method can be used to identify not only putative antiviral agents, but also cellular regulators of viral replication.     

Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA

Background and Aim:  We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro . In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo .
Methods:  We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo .
Results:  Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by ∼10⁻³. Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/ lox P switching system resulted in specific suppression of virus protein synthesis in the liver.
Conclusion:  Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.     

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.     

Up-regulation of Hedgehog pathway is associated with cellular permissiveness for hepatitis C virus replication

Studies of the hepatitis C virus (HCV) life-cycle rely heavily on Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh-related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here we demonstrate that Huh7.5 cells, when compared with Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers, and markedly up-regulated Hh pathway activity: Shh, >100-fold, Gli1, >30-fold, Ptc, 2-fold. In Huh7.5 cells, we found that cyclopamine, an Hh pathway antagonist, reduced HCV RNA levels by 50% compared with vehicle and inactive isomer controls. Moreover, in Huh7 cells treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2-fold and 4-fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose-response manner. CONCLUSION: We have identified a relationship between HCV and Hh signaling where up-regulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high-level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.     

Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection

Detection of residual HCV in individuals with SVR after treatment of CHC can be significantly heightened by analyzing ex vivo mitogen-activated T and B lymphocytes and applying sensitive nucleic acid amplification assays. However, it remained unknown if synergistic activation of lymphocytes and monocytes would further augment HCV detection, if viral replication becomes universally upregulated in treated cells, and if examining sequential sera and lymphoid cells would improve detection of occult infection. Using paired sera and lymphoid cells collected 1 year apart from 17 individuals with normal liver enzymes for up to 72 months after SVR, it was found that simultaneous activation of lymphocytes and monocytes enhanced identification of silent HCV infection and revealed that in some cases monocytes were the principal immune cell type where HCV persisted. Testing of serial samples further increased detection of occult infection. Ultimately, by combining the above two approaches, all individuals with SVR were found to be silent carriers of HCV. Clonal sequencing revealed HCV variations in sera and lymphoid cells and evolution of viral genomes confirming ongoing virus replication. Surprisingly, similar to those with CHC, naive lymphoid cells from some individuals carried approximately 10(3) HCV copies/microg total RNA. HCV loads in naive lymphoid cells predetermined the outcome of ex vivo stimulation with respect to upregulation or inhibition of HCV replication. HCV RNA levels in occult infection were inversely proportional to the expression of IFNalpha and IFN-inducible MxA, but not to IFNgamma or tumour necrosis factor alpha in naive and mitogen-treated lymphoid cells.